This invention relates to an apparatus and method for the structural elucidation and determination of small volumes of an analyte. More particularly, this invention relates to an apparatus and method for the structural elucidation and determination of analytes in nanoliter volume samples using a micro-nuclear magnetic resonance spectrometer (xcexcNMR).
Nuclear magnetic resonance spectroscopy, or NMR, is one of the most powerful and commonly used methods for the analysis and elucidation of the chemical structure of molecules. However, NMR suffers from an inherently low sensitivity. This low sensitivity is of particular concern when examining small sample volumes ( less than 1 ml), where the dependence of the NMR signal strength on sample volume results in an enormous reduction in the signal-to-noise ratio (SNR). A poor SNR is a fundamental limitation of NMR microspectroscopy. Conventional NMR spectrometers use radio frequency coils ranging from several millimeters to tens of centimeters in diameter to tightly couple to sample volumes that range from several microliters to greater than 1 liter. In addition, NMR spectroscopy requires that the high strength static magnetic field (B0) into which the sample is immersed be highly homogeneous ( less than 1 ppm over then entire volume of the sample) and necessitates the use of physically large, highly sophisticated, expensive superconducting magnets. Although recent advancements in high-field magnet technology have provided higher strength magnetic fields with improved homogeneity, the costly purchase price of these large magnets has precluded the development of customized NMR systems.
The radiofrequency (RF) coil used to receive the free-induction decay signal from the sample is a key component of the NMR spectrometer and has a profound effect on the observed SNR. In general, the RF coil can be used both to transmit energy to the sample thereby exciting the sample from its equilibrium state to its excited state, and to receive energy from the sample as it relaxes from its excited state to its equilibrium state. To optimize the detection efficiency, high performance coils with low resistivity and high inductance are designed to tightly couple to the sample and to present a highly homogeneous RF magnetic field to the sample. Although the vast majority of conventional NMR spectrometers use relatively large RF coils (mm to cm size) and samples in the xcexcl to ml volume range, there are significant performance advantages achieved by using smaller size coils when examining very small samples.
Unlike larger systems, where the dominant source of noise is the conducting sample, the primary noise in NMR spectroscopy of small samples is the thermal noise (also called the Johnson noise) of the RF coil. For example, when considering samples with conductivities similar to that of biological tissue (i.e., saline) and static magnetic field strengths of several Tesla, the transition from sample dominated noise to coil dominated noise occurs at a size scale of several millimeters. As the detection sensitivity of the RF coil increases inversely with coil diameter and the variation in coil resistance with coil size is less pronounced, the mass (detection) sensitivity of the system is enhanced at smaller dimensions. This has been the justification of several studies using microcoils to examine mass-limited or volume-limited samples. However, in all previous work, these coils have been used in conventional (large) NMR magnets and with conventional NMR spectrometers, and thus one Of the most significant advantages of microcoils has not been realized.
The present invention relates to xcexcNMR spectrometers using miniaturized and customized NMR magnets tailored to the smaller sizes and applications of micron to millimeter sized RF microcoils. By restricting the size of the sample to microscopic domain applications, the required volume over which the static magnetic field must be uniform is relaxed by several orders of magnitude. In addition, the working distance required between the pole faces (or opposing coils) of the magnet is significantly reduced when compared with conventional NMR systems. Consequently, when considering a microcoil-based, dedicated NMR spectrometer for investigations of mass and/or volume limited samples, and applications, that require reduced-scale geometries (e.g., capillary electrophoresis), the physical size of the magnet can be greatly reduced and the homogeneity constraints (i.e., the volume over which the magnetic field must be uniform) are significantly relaxed. An additional advantage of using a smaller magnet is that space allocation restrictions (due primarily to the stray magnetic lines of force that extend outside the magnet) are significantly reduced without the need of elaborate self-shielding designs.
Although the static field magnet represents the single most substantial contribution to the cost of the system, a xcexcNMR spectrometer specifically tailored to microdomain applications would benefit from reduced cost in other system components as well, (e.g., the RF power amplifier). The greatly reduced cost of such a system would make possible the acquisition of xcexcNMR spectrometers by research and educational groups interested in a narrower range of NMR applications, and consequently, the complexity of the computer and data acquisition components can be reduced to include a subset of available pulse sequences and detection and reconstruction algorithms deemed appropriate for the specific application and optimized for experimental conditions. Hence, chip-based (e.g., erasable programmable read only memory, EPROM) modules can be used to replace larger, more comprehensive front-end computer and data acquisition systems currently used in conventional NMR spectrometers, and would result in further cost reduction, potentially with an increase in performance for the specific application. Physician""s offices, educational classrooms, chemistry laboratories, and research and manufacturing sites where delicate or fragile products cannot be transported (e.g., a semiconductor manufacturing clean-room) are but a few of the locations where a low-cost, tabletop xcexcNMR spectrometer can be advantageous.
Finally, the reduced size of the xcexcNMR spectrometer provides a unique aspect of modularity. A natural extension of this aspect of the invention is to use NMR detection in conjunction with other investigative modalities and instrumentation (e.g., cell flow cytometry) as an add-on module. Although other detection schemes (e.g., laser-induced florescence) have previously been used in such capacities, the cost and size of NMR spectrometers have precluded the use of NMR in such applications.
Thus, the major advantages of the present xcexcNMR instrument include, but are not limited to, smaller size, lower cost, higher mass sensitivity compared to conventional NMR, and the ability to tailor such instruments to a particular application such as microseparations for even further improvement in performance and modularity, specifically to provide NMR detection and analysis capabilities to complement other more traditional detection modalities on existing research and analytical instrumentation.
Specifically, the present invention provides an NMR apparatus for analyzing and elucidating the chemical structure of an analyte sample, the apparatus comprising an analyte sample holder having a containment region that holds a volume of less than about 10 microliters of the analyte sample, a microcoil, which encloses the containment region of the analyte sample holder and the analyte sample contained therein, the microcoil having an inside dimension of less than about 1 mm, and the microcoil operatively associated with the analyte sample contained in the containment region of the analyte sample holder such that the microcoil can transmit and/or receive energy from the analyte sample in the containment region of the analyte sample holder, and a magnet having a mass less than about 50 kg, the magnet positioned about the microcoil and the analyte sample in the containment region of the analyte sample holder to provide a static magnetic field in which the microcoil and the analyte sample in the containment region of the analyte sample holder are located, wherein the microcoil and the magnet provide for the obtainment of an NMR spectrum of the analyte sample in the containment region of the analyte sample holder having a spectral line width resolution of less than about 0.1 parts per million.
The present invention also provides a method of analysis and elucidation of the chemical structure of an analyte using NMR, the method comprising providing an analyte sample that has a volume of less than about 10 microliters, enclosing the analyte sample in a microcoil wherein the inside dimension of the microcoil is less than about 1 mm, generating a static magnetic field about the analyte sample using a magnet where the mass of the magnet is less than about 50 kg, transmitting energy to the analyte sample to excite the sample from its equilibrium state to its excited state, detecting the energy emitted from the analyte sample when the sample relaxes from its excited state to its equilibrium state, and transforming the detected energy into a NMR spectrum having a spectral line width resolution of less than about 0.1 parts per million.